Subunit mutations affect ethanol actions on GABA(A) receptors expressed inXenopus oocytes

1 Mutations of specific amino acids were introduced in transmembrane domain s (TM) of GABA(A) receptor alpha(2), beta(1) and gamma(2L) subunits. The ef fects of these mutations on the action of ethanol were studied using the Xe nopus oocyte expression system and two-electrode voltage-clamp recording te chniques. 2 Mutant alpha(2) subunits containing S270I (TM2) or A291W (TM3) made the r eceptor more sensitive to GABA, as compared to wild-type alpha(2)beta(1)gam ma(2L) receptor. The mutation S265I (TM2) of beta(1) and S280I (TM2) or S30 1W (TM3) in gamma(2L) subunits did not alter apparent affinity of the recep tor for GABA. M286W (TM3) in the beta(1) subunit resulted in a receptor tha t was tonically open. 3 Using an ECS concentration of GABA, the function of the wild-type recepto r with alpha(2)beta(1)gamma(2L) subunits was potentiated by ethanol (50 - 2 00 mM). The mutations in TM2 or TM3 of the at subunit diminished the potent iation by ethanol. The action of ethanol was also eliminated with a mutatio n in the TM2 site of the beta(1) subunit. Ethanol produced significant inhi bition of GABA responses in receptors containing the combination of alpha(2 ) and beta(1) TM2 mutants with a wild-type gamma(2L) subunit. A small but s ignificant reduction in the potentiation by ethanol was observed with gamma (2L) TM2 and/or TM3 mutants. 4 From these results, we suggest that in heteromeric GABA(A) receptors comp osed of the alpha, beta and gamma subunits, ethanol may bind in a cavity fo rmed by TM2 and TM3, and that binding to the alpha or beta subunit may be m ore critical than the gamma subunit.